Models to study hepatitis C virus (HCV) in the laboratory have improved dramatically over the last decade with the advent of HCV cell culture infectious viruses, but these systems still produce relatively low viral titers. We attempted to improve viral titers by serially passaging the widely used laboratory adapted virus, J6/JFH-1 JC1 (JC1), under increasingly stringent conditions for 20 weeks, and the resulting virus produced titers 10 times higher than JC1. We designated this virus J6/JFH-1 JC1.1 (JC1.1). Sequence analysis and functional mapping of JC1.1 indicated the increase in titer required two amino acid substitutions in the NS3 protease and a single amino acid change at the amino-terminus of NS5B, the P1' position of the NS5A/NS5B cleavage site in the viral polyprotein. As these results hinted that JC1.1 might exhibit altered polyprotein cleavage kinetics, we used a pulse chase labeling strategy and observed that JC1.1, but not JC1, produced a small amount of relatively stable uncleaved NS5A-NS5B during infections. We developed a plasmid based overexpression system to supply the JC1.1 mutant NS5A-NS5B in trans during infections, thinking that the presence of more uncleaved NS5A-NS5B might result in even greater virus titers than we observed in JC1.1 infections. Surprisingly, we observed a 100-fold increase in titer over the original JC1 parental virus when the NS5A-NS5B from JC1.1 was supplied in trans to both JC1 and JC1.1 infections, indicating that uncleaved NS5A-NS5B has a dramatic impact on infectious virus production. We hypothesized that for a role in virus production, NS5A-NS5B should localize to the site of virion assembly and increases virion production and secretion in a manner dependent on the known assembly and membrane binding activities of the component proteins NS5A and NS5B. To test this hypothesis we developed a series of experiments to investigate the impact of NS5A-NS5B on virus assembly, RNA replication, particle secretion, specific infectivity, virus maturation and LDL association, and virion stability. We also wanted to understand how varying the processing efficiency of NS5A-NS5B correlated with virus production, so we generated a series of mutations in the P1 and P1' positions of NS5A-NS5B that produce a range of uncleaved NS5A-NS5B amounts and we propose to test them for cleavage efficiency and infectivity release. We also generated a panel of well-characterized mutations in NS5A and NS5B known to alter the assembly, replication, and membrane association functions of these proteins in their mature, fully processed forms, and we will assess how they impact virus production when supplied in the context of uncleaved NS5A-NS5B in trans in infections, with the hope that this will provide some insights into how NS5A- NS5B enhances virus production. We also have designed approaches to determine where NS5A-NS5B localizes in infected cells with a focus on sites of viral RNA replication and virion biogenesis. Completing our aims will define how and where NS5A-NS5B functions in virion production, highlight the novel role of uncleaved polyprotein intermediates in the HCV lifecycle, and potentially define a novel antiviral target.